Abstract: Objective To investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro. Methods HepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy. Results Compared with the control group, the cells exposed to2 μmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G0/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G0/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm. Conclusion DHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.